Optical measurement of neuronal activity in the developing cerebellum of zebrafish using voltage-sensitive dye imaging.

Voltage-sensitive dye (VSD) imaging enables fast, direct, and simultaneous detection of membrane potentials from a population of neurons forming neuronal circuits. This enables the detection of hyperpolarization together with depolarization, whose balance plays a pivotal role in the function of many brain regions. Among these is the cerebellum, which contains a significant number of inhibitory neurons. However, the mechanism underlying the functional development remains unclear. In this study, we used a model system ideal to study neurogenesis by applying VSD imaging to the cerebellum of zebrafish larvae to analyze the neuronal activity of the developing cerebellum, focusing on both excitation and inhibition. We performed in-vivo high-speed imaging of the entire cerebellum of the zebrafish, which was stained using Di-4-ANEPPS, a widely used VSD. To examine whether neuronal activity in the zebrafish cerebellum could be detected by this VSD, we applied electrical stimulation during VSD imaging, which showed that depolarization was detected widely in the cerebellum upon stimulation. These responses mostly disappeared following treatment with tetrodotoxin, indicating that Di-4-ANEPPS enabled optical measurement of neuronal activity in the developing cerebellum of zebrafish. Moreover, hyperpolarizing signals were also detected upon stimulation, but these were significantly reduced by treatment with picrotoxin, a GABAA receptor inhibitor, indicating that these responses represent inhibitory signals. This approach will enable a detailed analysis of the spatiotemporal dynamics of the excitation and inhibition in the cerebellum along its developmental stages, leading to a deeper understanding of the functional development of the cerebellum in vertebrates.